This disclosure relates to sensors for analyte detection and to methods of manufacture thereof. More specifically, this disclosure relates to biosensors and to methods of detection of biological metabolites and other analytes.
Numerous clinical trials and intensive research efforts have indicated that continuous metabolic monitoring holds great potential to provide an early indication of various body disorders and diseases. In view of this, the development of biosensors for the measurement of metabolites has become an area of significant scientific and technological study for various research groups across the world. A useful class of biosensors are electrochemical sensors that link enzymatic reactions to electroactive products. These sensors also enable the detection of small volumes of bio-analytes in clinical or home use applications. For example, the development of miniaturized implantable sensors for continuous monitoring of glucose is useful for optimal care of diabetes mellitus. Many other clinical situations also necessitate the measurement of various body metabolites like lactate, creatinine, creatine, glutamate, phosphate, cysteine, homocysteine, and the like. For example, a device that can measure lactate levels has important implications in a number of diseases and conditions (e.g., to indicate muscle fatigue, shock, sepsis, kidney disorders, liver disorders and congested heart failure). In some clinical situations, simultaneous monitoring of two or more metabolites is desirable.
For example, the complex interrelationship between glucose and other metabolic analytes induces one to simultaneously detect glucose, glutamate, lactate, oxygen, carbon dioxide, and the like. Simultaneous monitoring of brain glucose, lactate and oxygen gives a comprehensive picture of complementary energy supply to the brain in response to acute neuronal activation. Levels of glucose and glutamate in cerebrospinal fluid are important in the control of diseases such as meningitis.
Currently, most of the electrochemical sensors used for the specific detection of lactate, glucose, glutamate, and the like, employ analyte specific enzymes, and are based on the Clark-type amperometric detection. For example, first generation Clark-type glucose sensors employ the glucose oxidase enzyme (GOx), immobilized on top of a working electrode. This enzyme catalyses the oxidation of glucose to glucarolactone, as shown in reaction (1).
                              Glucose          +                      O            2                          ⁢                  →                      glu            ⁢                                                  ⁢            cos            ⁢                                                  ⁢            eoxidase                          ⁢                  Glucarolactone          +                                    H              2                        ⁢                          O              2                                                          (        1        )            
The generated H2O2 is amperometrically assessed on the surface of a working electrode according to reaction (2) shown below.
                                          H            2                    ⁢                      O            2                          ⁢                  →          V                ⁢                              O            2                    +                      2            ⁢                          H              +                                +                      2            ⁢                          e              -                                                          (        2        )            
Currently, these biosensors suffer from two major pitfalls: (i) lack of miniaturization compatible with roll-to-roll production and (ii) lack of high sensor performance that is desirable for most of the in vivo applications.
The desire for miniaturization occurs from the complex applications these biosensors are being utilized for. Typical applications for these in vivo biosensors include metabolite monitoring in the neurons, extra cellular space, eyes, subcutaneous (s.c.) tissue, veins, and the like. In some cases, the biosensor is used in the affected area of the particular organ to diagnose the ailment and is left in place to monitor the condition of the ailment. In all of these applications, it is desirable to have miniaturization in order to avoid damage to the healthy tissue and to reduce wound recovery time, infection and patient discomfort.
Miniaturization of biosensors and sensor performance are two diametrically opposed issues. For example, most of the current miniaturization strategies result in a decrease in sensor performance as a result of reduced active working area, reduced enzyme loading and reduced signal-to-noise ratio. Any increase in the size of the biosensor will cause a large damage to the local tissue that will augment the magnitude of a foreign body response. This foreign body response will further decrease sensor performance by decreasing the analyte flux and possibly denaturing the sensing enzymes.
To alleviate some of the aforementioned issues of miniaturization, a number of micro-sensory devices based on microelectromechanical systems (MEMS) technology have been reported with advantages such as high precision, high functionality and mass-production. However, this technology uses expensive equipment and materials that could lead to an overall increase in the cost per piece of the biosensor. Moreover, these devices are based on inflexible or brittle materials such as silicon and glass, which have a higher chance of breakage within the in vivo environment.
In order to simultaneously afford miniaturization while at the same time increasing enzyme loading (to improve sensor performance), a silicon micro-machined needle-shaped structure for glucose monitoring has been reported. These needle-shaped biosensors along with channels for fluid flow and enzyme housing are created by wet and dry etching processes, while the (Ti/Pt) titanium/platinum working and (Ag/AgCl) silver/silver chloride reference electrodes located at the tip of the needle-shaped biosensors are patterned by photolithography. However as mentioned above, since these devices are made up of silicon substrates, these are more prone to breakage within the body.
In order to avoid the problem of sensor breakage in the body, biosensors have been fabricated, where the sensing electrodes are patterned on a polymeric KAPTON® film and subsequently rolled up to form a two dimensional cylindrical electrode. While the soft and flexible nature of these sensors presents an advantage over currently available techniques, these sensors are not reproducible on large scale and have a large sensor-to-sensor variability because of the large effect of the roll-up angle on the performance of the sensor. Moreover, the problem of enzyme loading and low electroactive surface still persists.
Biosensor miniaturization based on electrodes patterned on planar (both rigid as well as flexible) substrates have also been reported. However, these planar sensors do not afford 3-D analyte diffusion that is desirable for enhanced sensor performance. For enhanced sensor performance, miniaturized sensors based on micro-disc array electrodes have been reported, which use expensive machinery and cannot be easily produced at lower cost. Furthermore, these have problems of low electroactive area, lack of reusable electrodes and reduced enzyme loading.
Various reports have also emerged on the use of electrodes decorated with nanostructured materials such as nanoparticles, nanotubes and nanocubes to enhance the electroactive areas and enzyme loading. These nanostructured materials tend to be fouled very quick, thereby resulting in a quick loss of sensor performance.
Based on the above, it is desirable to develop methodologies for the production of biosensors that simultaneously afford extreme miniaturization, high sensor performance and flexibility for roll-to-roll production. Such methodologies will improve the quality of point of care diagnosis and prognosis of various body disorders.